Upon exposure to Entamoeba histolytica antigen, lymphocytes from patients treated for amebic liver abscess produce lymphokines which activate monocyte-derived macrophages to kill E. histolytica trophozoites. We now demonstrate that gamma interferon (IFN-γ) is produced by these stimulated lymphocytes and is sufficient but not exclusively necessary to activate monocyte-derived macrophage amebicidal activity. Supernatants from mononuclear cells of 7 patients when stimulated with amebic antigen contained more IFN-γ than comparable supernatants derived from control cells (1,862 U/ml vs. 174 U/ml geometric means, P < 0.01); IFN-γ levels were similar in patient and control supernatants following concanavalin A stimulation. Macrophages activated solely by partially purified IFN-γ or recombinant human IFN-γ (300 U/ml) killed 47% of virulent amebae by 6 hr at 37°C. Monocyte-derived macrophages stimulated with lymphokines elicited by amebic antigen or concanavalin A killed 48% and 57% of axenic E. histolytica trophozoites, respectively, over 6 hr at 37°C (P < 0.001 for each compared to control). Macrophages incubated with the identical lymphokines, but in the presence of monoclonal antibody to IFN-γ, were only able to kill 18% and 27% of amebae, respectively, at 6 hr (P < 0.05 to control or when antibody to IFN-γ was not present). If antibody to IFN-γ was added to the stimulating lymphokine, more macrophages died during interaction with amebae (P < 0.05). In summary, IFN-γ has a major but not exclusive role in activating human monocyte-derived macrophages in vitro to kill virulent E. histolytica trophozoites.